JP6840332B2 - Immunostimulatory oligonucleotide complex - Google Patents

Description

The present invention relates to immunostimulatory oligonucleotide complexes, in particular a double-stranded oligonucleotide and a carrier complexed with an I such as interferon-α (IFN-α) or interferon-β (IFN-β). It relates to a complex having excellent inducing activity of cytokines such as interferon type and interleukin-12 (IL-12) and acting as an adjuvant.

Innate immunity is the body's initial defense response to microbial infection. The constituents of the microorganism are Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), DNA-dependent activator of IFN-regulatory factors (DAI), IFN-γ-inducible protein. It provokes various immune responses by being recognized by receptors such as 16 (IFI16), DDX41, and cyclic GMP-AMP synthase (cGAS).

Toll-like receptors (TLRs) form homodimers or heterodimers by binding ligands on single-transmembrane receptors and transmit signals. There are 10 types in humans, of which TLR3 (recognizing double-stranded RNA), TLR7 and TLR8 (recognizing single-strand RNA), and TLR9 (recognizing unmethylated CpG DNA) recognize nucleic acids. Is. TLR3, TLR7, TLR8 and TLR9 are mainly localized in intracellular organelle membranes such as ER and endosomes, and endosomes recognize ligands and transmit signals. On the other hand, TLR1 (recognizing triacyl lipoprotein, etc.), TLR2 (recognizing peptidoglycan, etc.), TLR4 (recognizing lipopolysaccharide, etc.), TLR5 (recognizing flagellin, etc.) that recognizes sugars, lipids, and proteins derived from bacteria and viruses. , TLR6 (recognizing diacyl lipoproteins, etc.) are all present on the cell surface, and recognize the surface layer components of bacteria on the cell surface and transmit signals.

NOD-like receptors (NLRs) are intracellular receptors that form a large family of over 30. It specifically recognizes peptidoglycan, which is mainly derived from microorganisms.
RIG-I-like receptors (RLRs) are a family of intracellular RNA helicases that recognize RNA present in the cytoplasm.
DNA-dependent activator of IFN-regulatory factors (DAI), IFN-γ-inducible protein 16 (IFI16), DDX41, cyclic GMP-AMP synthase (cGAS) have been identified as intracellular DNA receptors, but also cytoplasm. The presence of internal DNA receptors has been suggested.

As described above, various receptors are individually or jointly involved in immune activity, and the whole picture has not been elucidated.

Infectious diseases, cancer, allergies, etc. are improved by enhancing immune activity. For these diseases, research and development using synthesized oligodeoxynucleotides containing unmethylated cytosine-guanine sequence (CpG) (hereinafter sometimes referred to as "ODN") as immunostimulatory drugs has been conducted since 2000. It is actively done. The CpG ODN developed so far is roughly classified into three classes (class A, class B, and class C).

Class A (also called D type) CpG-A ODN contains CpG in the palindromic sequence of the phosphodiester skeleton, with the polyguanine sequence of the phosphorothioate skeleton added to both ends (3'and 5'ends). Chain ODN (eg, ODN2216, ODN1585, D35 shown in Table 1). CpG-A ODN has the property that two molecules complementarily form a double strand by the parindrome sequence of the phosphodiester skeleton, and the polyguanine sequences at both ends form a guanine tetramer. The main chain CpG-A ODN further self-associates to form a tetramer. This tetramer further self-associates with a double-stranded CpG-A ODN or a single-stranded CpG-A ODN molecule to form a higher-order structure (Non-Patent Document 1). CpG-A ODN is mainly recognized by TLR9 in dendritic cells and induces type I interferon (IFN) such as interferon-α (IFN-α) and interferon-β (IFN-β) (Patent Document 1, non-). Patent Document 2).

Class B (also called K type) CpG-B ODN is a single-stranded ODN consisting only of a phosphorothioate backbone containing CpG (eg, ODN1826, ODN2006, K3 shown in Table 1). CpG-B ODN is recognized primarily by B cell TLR9 and induces inflammatory cytokines such as interleukin-6 (IL-6) and IL-12 or tumor necrosis factors (TNFs) (non-patented). Document 3). It is known that when CpG-B ODN is electrostatically bound to a cationic peptide or a cationic liposome to form a complex, type I IFN is induced in the same manner as CpG-A (Non-Patent Document 4). ).

Class C CpG-C ODN is a single-stranded ODN consisting only of a phosphorothioate backbone containing a palindromic sequence on the 3'terminal side and contains CpG on both the 5'terminal side and the 3'terminal side (eg,). ODN2395 shown in Table 1). In CpG-C ODN, two molecules form a partial double strand due to the palindromic sequence. CpG-C ODN exhibits intermediate properties between CpG-A and CpG-B and induces both inflammatory cytokines and type I IFN (Non-Patent Document 5).

Japanese Unexamined Patent Publication No. 2008-516634

Klein et al. Higher order structure of short immunostimulant oligonucleotides studies by atomic force microscopy. Ultramicroscopy, 110: 689-693 (2010) Krug et al. Identification of CpG oligonucleotide sequences with high induction of IFN-alpha / beta in plasmacytoid dendritic cells. Eur. J. Immunol. 31: 2154-2163 (2001) Hartmann et al. Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo. J. Immunol. 164: 1617-1624 (2000) Gungor B., Yagci FC., Tincer G., Bayyurt B., Alpdundar E., Yildiz S., Ozcan M., Gurcel I., Gurcel M. CpG ODN nanorings induce IFNα from plasmacytoid dendritic cells and demonstrate potent vaccine adjuvant activity .Science Translational Medicine 6, 235ra61 (2014) Poeck et al. Plasmacytoid dendritic cells, antigen, and CpG-C license human B cells for plasma cell differentiation and immunoglobulin production in the absence of T-cell help. Blood, 103: 3058-3064 (2004)

In these conventional CpG ODNs, all or part of the ODN skeleton is phosphorothioated, that is, the oxygen atom of any of the phosphate groups of the nucleotides constituting the ODN is replaced by a sulfur atom. ODNs having a phosphorothioate skeleton become resistant to DNA-degrading enzymes in the body, and their uptake efficiency into cells is improved as compared with ODNs consisting only of a phosphodiester skeleton. However, it has been pointed out that ODN having a phosphorothioate skeleton binds non-specifically to a protein, and there is a concern about side effects due to it.

Further, CpG-A ODN has a higher-order structure as described above, and the structure is a form in which various higher-order structures naturally generated in the process of synthesis are mixed. It can be said that it is impossible to specify a higher-order structure showing a high IFN-inducing effect, let alone industrially control this so that a specific higher-order structure is formed, and this is CpG-A. It hinders the clinical application of ODN. The above-mentioned Non-Patent Document 4 proposes to prepare a uniform nanocyclic structure by complexing CpG-B with a cationic peptide, but the number of bases of ODN, the ratio with a carrier, etc. are limited. Lack of design freedom.

Therefore, an object of the present invention is to provide an immunostimulating oligonucleotide which is excellent in type I IFN-inducing activity even if it is not phosphorothioated and is suitable for industrial production.

As a result of various studies to achieve the above objectives, surprisingly, a single-stranded ODN containing CpG and not phosphorothioated was converted into a linear double-stranded ODN, and the double-stranded ODN was complexed with a carrier. , TLR9 was recognized to induce type I IFN, and the present invention was completed. That is, the present invention is as follows:

[1] A linear double-stranded oligonucleotide containing 10 to 100 base pairs, and each single-stranded oligonucleotide constituting the double strand has a phosphodiester-mediated citocin-guanine sequence (CpG). Double-stranded oligonucleotides containing 2 to 20 and in which 90% or more of the nucleotide-to-nucleotide bonds of each single-stranded oligonucleotide are phosphodiester bonds;
[2] The double-stranded oligonucleotide according to [1], wherein each of the single-stranded oligonucleotides does not contain a palindrome sequence;
[3] The double-stranded oligonucleotide according to [1] or [2], wherein the bonds between the nucleotides of each single-stranded oligonucleotide are all phosphodiester bonds;
[4] Any of [1] to [3], wherein the single-stranded oligonucleotide has the following base sequence and any sequence in which 1 to 3 bases other than CpG are deleted, substituted or added in the sequence. The double-stranded oligonucleotide according to item 1.
5'-TCGTCGTTTTGTCGTTTTGTCGTT-3'(SEQ ID NO: 1)
5'-TCGTCGTTTTGTCGTTTTGTCGTTTCGTCGTTTTGTCGTTTTGTCGTT-3'(SEQ ID NO: 2)
5'-TCGTCGTTTTGTCGTTTTGTCGTTTCGTCGTTTTGTCGTTTTGTCGTTTCGTCGTTTTGTCGTTTTGTCGTT-3'(SEQ ID NO: 3)
5'-ATCGACTCTCGAGCGTTCTC-3' (SEQ ID NO: 4);
[5] An immunostimulatory oligonucleotide complex comprising a carrier and the double-stranded oligonucleotide according to any one of [1] to [4] complexed with the carrier;
[6] The immunostimulating oligonucleotide complex according to [5], wherein the carrier is selected from liposomes, polymeric compounds, and inorganic compounds;
[7] The immunostimulating oligonucleotide complex according to [5] or [6], wherein the average particle size is 100 nm or more, preferably 250 nm or more, more preferably 700 nm or more;
[8] The weight ratio of the double-stranded oligonucleotide to the carrier is 0.05: 1 to 10: 1, preferably 0.1: 1 to 10: 1, and more preferably 0.15: 1 to 10: 1. There is an immunostimulating oligonucleotide complex according to [5] or [6];
[9] A vaccine adjuvant comprising the immunostimulatory oligonucleotide complex according to any one of [5] to [8].

[10] A method for preventing infectious diseases of mammals including humans, birds or fish, which is a detoxified or attenuated antigen derived from a pathogen causing the infectious disease, and [5] to [8]. The method comprising continuously or simultaneously administering the immunostimulatory oligonucleotide complex according to any one of the above items to promote the production of antibodies against pathogens in the body and acquire immunity against infectious diseases;
[11] Use of the immunostimulatory oligonucleotide complex according to any one of [5] to [8] in the production of a vaccine for preventing infectious diseases in mammals including humans, birds or fish;
[12] Use of the immunostimulatory oligonucleotide complex according to any one of [5] to [8] for the prevention of infectious diseases in mammals including humans, birds or fish.

[13] A method for treating or preventing cancer.
By continuously or simultaneously administering the cancer antigen or a part thereof and the immunostimulatory oligonucleotide complex according to any one of [5] to [8], cytotoxic T cells against the cancer antigen are administered into the body. A method of treating or preventing cancer by inducing cells (CTL) and attacking cancer cells that present cancer antigens;
[14] Use of the immunostimulatory oligonucleotide complex according to any one of [5] to [8] in the production of a vaccine for treating or preventing cancer;
[15] Use of the immunostimulatory oligonucleotide complex according to any one of [5] to [8] for the treatment or prevention of cancer.

[16] A pharmaceutical composition for treating or preventing allergies, which comprises the immunostimulating oligonucleotide complex according to any one of [5] to [8];
[17] The pharmaceutical composition according to [16], which further comprises an allergen or a part thereof.

[18] A method for treating or preventing allergies.
To activate allergen-specific helper 1T (Th1) cells more than helper 2T (Th2) cells by administering the immunostimulatory oligonucleotide complex according to any one of [5] to [8]. How to treat or prevent allergies;
[19] The method of [18], further comprising administering the allergen or a portion thereof continuously or simultaneously with the immunostimulating oligonucleotide complex;
[20] Use of the immunostimulatory oligonucleotide complex according to any one of [5] to [8] in the manufacture of a pharmaceutical for allergy treatment or prevention;
[21] Use of the immunostimulatory oligonucleotide complex according to any one of [5] to [8] for the treatment or prevention of allergies.

In the double-stranded oligonucleotide of the present invention, 90% or more of the bonds between nucleotides are phosphodiester bonds, and substantially no phosphorothioate bond is contained, so that there is no concern about side effects. In addition, complexation is easy, and the resulting complex has a controlled structure and size, and is expected to be applied as an adjuvant. The complex is recognized by TLR9 in antigen-presenting cells and mainly induces type I IFN, the amount of which is significantly higher than when CpG-B ODN is supported on the same carrier in the same way.

FIG. 1 is a graph showing the induction (relative expression level) of IFN-β from mouse macrophage-like cells shown in Example 1. The CpG ODN complex of the present invention, B24-PD and lipofectamine 2000, showed high IFN-β inducibility at the double strand. *, p <0.05 FIG. 2 is a graph showing the induction of IFN-β from mouse macrophage-like cells shown in Example 2. The double-stranded CpG ODN complex of the present invention showed high IFN-β inducibility at 48 base pairs or more. *, p <0.05 FIG. 3 is a graph showing the induction of IL-12 from mouse macrophage-like cells shown in Example 3. It was shown that the double-stranded CpG ODN complex of the present invention retains not only IFN-β but also IL-12 inducibility. *, p <0.05 FIG. 4 shows the induction of IFN-β from mouse macrophage-like cells shown in Example 4. It was shown that complexing with a carrier is essential for the induction of IFN-β from the phosphodiester double-stranded linear CpG ODN of the present invention. *, p <0.05 FIG. 5 shows the induction of IFN-β from mouse macrophage-like cells shown in Example 5. It was shown that the double-stranded CpG ODN complex of the present invention also induces IFN-β in cationic liposomes other than Lipofectamine 2000. *, p <0.05 FIG. 6 shows a comparison of the IFN-β induction amount of the complex with lipofectamine 2000 shown in FIG. 4 and the complex with DOTAP shown in FIG. The double-stranded CpG ODN of the present invention showed higher IFN-β inducibility when combined with Lipofectamine 2000 than when combined with DOTAP. *, p <0.05 FIG. 7 shows the induction of IFN-β from mouse macrophage-like cells shown in Example 6. The double-stranded CpG ODN complex of the present invention also showed high IFN-β inducibility in calcium phosphate particles. *, p <0.05 FIG. 8 shows the induction of IFN-α from human plasmacytoid dendritic cells, as shown in Example 7. When the double-stranded CpG ODN of the present invention was combined with DOTAP, it showed high IFN-α inducibility. *, p <0.05 FIG. 9 shows the induction of IL-6 from human B cells as shown in Example 7. It was shown that the phosphodiester double-stranded linear CpG ODN, both in the free state and in the complex with DOTAP, had a lower amount of IL-6 induced than the single-strand. It was also shown to be lower than single-stranded and double-stranded phosphorothioate CpG ODN. *, p <0.05 FIG. 10 shows the induction of IFN-α from human monocytes, as shown in Example 7. The double-stranded CpG ODN complex of the present invention was also recognized by the cytoplasmic DNA receptor and was shown to induce IFN-α. *, p <0.05 FIG. 11 is a graph showing the change in the size of Lipofectamine 2000 over time. Lipofectamine 2000 has been shown to form agglomerates over time. FIG. 12 is a graph showing the relationship between the complex size of ds B72-PD and lipofectamine 2000 and the amount of IFN-β induction. It was shown that the complex size is 700 nm or more and that it has a high ability to induce IFN-β in stimulation of mouse macrophage-like cells. FIG. 13 is a graph showing the effect of the amount of ds B72-PD bound to lipofectamine 2000 on IFN-β induction. When the concentration of lipofectamine 2000 in the culture medium is 5 μg / ml, if the binding amount of ds B72-PD is at least 0.5 or more based on the weight of lipofectamine 2000, the stimulation to mouse macrophage-like cells It was shown that high IFN-β inducibility was obtained. FIG. 14 is a graph showing the effect of the addition concentration of the complex of ds B72-PD and lipofectamine 2000 on IFN-β. It was shown that the amount of IFN-β induced from mouse macrophage-like cells depends on the amount of ds B72-PD and the lipofectamine 2000 complex added. FIG. 15 is a graph showing IFN-β induction from mouse macrophage-like cells by a complex of double-stranded CpG ODN and lipofectamine 2000 in which the sequence of ds B72-PD was partially modified. Both double-stranded CpG ODN (ds B72M1-PD, ds B72M2-PD, ds B72M3-PD, ds B72M4-PD, ds B72M5-PD) complexes have the same high IFN-β as the ds B72-PD complex. It showed inducibility. FIG. 16 is a graph showing IFN-β induction from mouse macrophage-like cells by a complex of double-stranded CpG ODN and DOTAP in which the sequence of ds B72-PD was partially modified. IFN-β induction higher in any double-stranded CpG ODN (ds B72M1-PD, ds B72M2-PD, ds B72M3-PD, ds B72M4-PD, ds B72M5-PD) complex than in the ds B72-PD complex Showed noh. FIG. 17 is a graph showing IFN-β induction from mouse macrophage-like cells by a complex of double-stranded CpG ODN and lipofectamine 2000 in which the sequence of ds B48-PD was partially modified. TLR9-mediated IFN-β induction in any double-stranded CpG ODN (ds 48M1-PD, dsB48M2-PD, ds B48M3-PD, ds B48M4-PD, ds B48M5-PD, ds CpG48-PD) complex Shown. FIG. 18 is a graph showing the inducing effect of OVA-specific CD8-positive T cells by the complex of ds B72-PD and lipofectamine 2000. Mean values of the proportion of OVA-specific CD8-positive T cells in blood CD8-positive T cells in 6 mice were shown to be significantly increased in the group receiving the ds B72-PD complex with OVA. Was done. **, p <0.05; NS, no significance FIG. 19 is a graph showing the effect of the complex of ds B72-PD and lipofectamine 2000 on the production of OVA-specific IgG1 antibody. Mean values of serum OVA-specific IgG1 antibody levels in 6 mice were shown to be significantly increased in the ds B72-PD complex with OVA compared to OVA alone. It was. **, p <0.05 FIG. 20 is a graph showing the effect of the complex of ds B72-PD and lipofectamine 2000 on the production of OVA-specific IgG2a antibody. Mean values of serum OVA-specific IgG2a antibody levels in 6 mice were shown to be significantly increased in the ds B72-PD complex with OVA compared to OVA alone. It was. **, p <0.05; NS, no significance FIG. 21 is a graph showing the effect of the complex of ds B72-PD and lipofectamine 2000 on the production of OVA-specific IgE antibody. Mean values of serum OVA-specific IgE antibody levels in 6 mice were shown to be increased in the group receiving the ds B72-PD complex with OVA, but not significantly. NS, no significance FIG. 22 is a graph showing the proportion of CD80-positive cells in human peripheral blood mononuclear cells. CD80 is one of the co-stimulators required for antigen presentation. The CD80 inducibility of the ds B72-PD complex was higher than that of the ds CpG-free72-PD complex, and its inducibility was similar to that of the CpG-A ODN2216 (A2216 in the figure) complex. FIG. 23 is a graph comparing the IFN-β inducibility of RAW264.7 cells with the complex of ds B72-PD and lipofectamine 2000 with the complex of plasmid vector pAcGFP-N1 and lipofectamine 2000. It was shown that the linear structure ds B72-PD complex has higher IFN-β inducibility than the cyclic structure plasmid vector complex. *, p <0.05 FIG. 24 is a graph comparing the IL-12 inducibility of RAW264.7 cells with the complex of ds B72-PD and lipofectamine 2000 with the complex of plasmid vector pAcGFP-N1 and lipofectamine 2000. It was shown that the linear structure ds B72-PD complex has higher IL-12 inducibility than the cyclic structure plasmid vector complex. *, p <0.05

<Double-stranded oligonucleotide>
In the present invention, the "oligonucleotide" is an oligonucleotide (ODN), which is a base selected from adenine (A), guanine (G), cytosine (C), and thymine (T) via deoxyribose. It consists of a plurality of structural units to which phosphoric acid is bound.

The double-stranded CpG ODN of the present invention contains 10 to 100 base pairs, preferably 20 to 80 base pairs. Each single-strand CpG ODN constituting the double-strand may have different base lengths from each other, or may not have a completely complementary base sequence. It is preferable that at least 3 bases of CpG and 3 bases before and after CpG are complementary. More preferably, the single-strand CpG ODNs have 80% or more complementarity, and even more preferably 90% or more complementarity. Most preferably, each single-stranded CpG ODN has the same base length, has 100% complementarity, and also forms double strands at the 3'and 5'ends.

The double-stranded CpG ODN is linear. A typical example of the linear structure is a structure in which the center line of the double helix structure is linear. However, in the present invention, "linear" means a cyclic structure in which both ends of the center line of the double helix structure are closed, for example, a double-stranded cyclic structure such as plasmid DNA and a sequence non-complementary to the single-stranded DNA are continuous. Includes a wide range of structures, excluding large loop structures or massive structures formed by doing so, such as the tetrameric structure of CpG-A ODN. Further, a part or an end of the linear structure may include a linear or small loop-shaped portion that does not form a double strand having a length of several bases to 10 bases.

Each single-strand CpG ODN contains 2 to 20, preferably 4 or more, more preferably 6 or more cytosine-guanine sequences (CpG) linked via a phosphodiester. Nucleotides other than CpG may be arbitrary. The position of CpG in the base sequence is not particularly limited, but is preferably 1 nucleotide or more away from the 3'and 5'ends. Further, it is preferable that CpGs are separated from each other by one or more nucleotides.

The bonds between nucleotides in each single-strand CpG ODN are 90% or more, preferably 95% or more, and most preferably 100% of the total number of bonds are phosphodiester bonds. Phosphodiester bonds include, for example, a phosphorothioate bond in which the oxygen atom of the phosphate group of the nucleotide is replaced with a sulfur atom, and a 2'-O, 4'-C-methano-bridged nucleic acid (2) in which the sugar of the nucleotide is modified. ', 4'-BNA) and its derivatives such as 3'-amino-BNA and 5'-amino-BNA structures may be contained in 10% or less of the total number of bonds.

Each single-strand CpG ODN has the effect of inducing type I IFN even if it does not contain a palindrome sequence, and preferably does not contain a palindrome sequence.

A preferred example of the single-stranded CpG ODN in the present invention has the following base sequence and any sequence in which 1 to 3 bases other than CpG are deleted, substituted or added.

5'-TCGTCGTTTTGTCGTTTTGTCGTT-3'(SEQ ID NO: 1)
5'-TCGTCGTTTTGTCGTTTTGTCGTTTCGTCGTTTTGTCGTTTTGTCGTT-3'(SEQ ID NO: 2)
5'-TCGTCGTTTTGTCGTTTTGTCGTTTCGTCGTTTTGTCGTTTTGTCGTTTCGTCGTTTTGTCGTTTTGTCGTT-3'(SEQ ID NO: 3)
5'-ATCGACTCTCGAGCGTTCTC-3' (SEQ ID NO: 4)

In the above sequence, all the bonds between nucleotides are phosphodiester bonds, but as described above, some of them may be phosphodiester bonds or the like. In addition, the other single-stranded CpG ODN has a sequence that is complementary to the above sequence. As described with respect to Table 1, the uppercase bases in the present specification are bound by phosphodiester bonds.

To prepare linear double-stranded CpG ODN, each single-stranded CpG ODN is synthesized separately by a nucleic acid synthesizer, then equimolarly mixed in a buffer, and at about 88 to about 98 ° C., about 5 to about. It can be obtained by heating for 30 minutes and then gradually lowering the temperature at about 0.1 to about 2 ° C./min. At this time, each single-stranded CpG ODN has a base sequence complementary to each other, and the complementary bases of each single-stranded CpG ODN form a hydrogen bond to form a double-stranded CpG ODN.
Another method for obtaining linear double-stranded CpG ODN is to amplify the region containing CpG by PCR using the bacterial or viral genome ODN as a template. Furthermore, the circular plasmid ODN can be amplified in the host cell, and the recovered circular plasmid can be cleaved with a restriction enzyme to obtain it.

<Immune-stimulated oligonucleotide complex>
A second aspect of the present invention is an immunostimulatory oligonucleotide complex in which the double-stranded CpG ODN prepared as described above and a physiologically acceptable carrier are complexed. By complexing, the type I IFN inducing ability becomes remarkable. "Physiologically acceptable carrier" refers to a substance that does not impair cells, tissues or organs in the body in a manner that impairs the object of the present invention. Examples of such carriers include, for example, polymeric compounds, emulsions, liposomes, inorganic compound particles, metal particles, metal oxide particles, carbon-based particles, and modifications thereof, preferably cationic. ..

Examples of the polymer compound include cationic polymers such as polyethyleneimine, chitosan, polylysine, LL-37, and Tat. These cationic polymers electrostatically bind to the double-stranded CpG ODN of the present invention. There is also a method of encapsulating DNA in poly (lactic-co-glycolic acid) (PLGA), which is a biodegradable polymer. In addition, a multimer such as a dendrimer can also be used as a carrier.

Examples of the emulsion include a water / oil type emulsion and a water / oil / water type emulsion, and there may be a method of including a double-stranded CpG ODN in the aqueous phase. Liposomes include double-stranded CpG ODN in a liposome composed of a lipid bilayer membrane, and cationic liposomes, for example, those containing a long-chain alkyl group and an amino group or an ammonium group. There is a method of electrostatically coupling the two.

Examples of the inorganic compound particles include calcium phosphate particles, hydroxide apatite particles, carbonic acid apatite particles, silica nanoparticles and the like. Examples of the metal particles include gold particles, silver particles, platinum particles, and silicon nanoparticles. Examples of the metal oxide particles include zinc oxide particles, titanium dioxide particles, alumina particles, and zirconia particles. Examples of carbon-based particles include fullerenes, carbon nanotubes, and carbon nanohorns.

The size of these carrier particles is not particularly limited, but the average length or longest diameter (D ₅₀ ) is preferably 10 nm to 1 μm, more preferably 100 to 800 nm. The particle shape is not particularly limited, and may be spherical, flake-shaped, columnar, or the like.

Among the above carriers, cationic liposomes and inorganic compound particles are preferable, and cationic liposomes containing a long-chain alkyl group and an amino group or an ammonium group, for example, Lipofectamine ™, DOTAP (N- [1-(). 2,3-Dioleoyloxy) propyl] -N, N, N-trimethylammonium methylsulfate), DMTAP (dimyristoyltrimethylammonium propane), DOAB (dimethyldioctadecylammonium (bromide salt)), DODAP (1,2-dioleoyl-3-dimethylammonium-propane), DC -CHOL (3b- [N', N'-dimethylaminoethane) -caramoyl] cholesterol hycrochloride), DOSPA (N- [2-[(1,5,10,14-Tetraazatetradecane-1-yl) carbonylamino] ethyl ] -N, N-dimethyl-2,3-bis (oleoyloxy) -1-propanaminium), etc., or a mixture with uncharged DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamin), etc. It may be (for example, DOSPA / DOPE (3: 1 wt / wt)), and calcium phosphate or DEAE dextran is preferable as the inorganic compound. Alternatively, a mixture thereof is preferable.

The method for complexing the double-stranded CpG ODN of the present invention with these carriers is not particularly limited. A carrier whose surface is positively charged can be electrostatically bound by mixing double-stranded CpG ODN in a suitable buffer. Double-stranded CpG ODN is adsorbed on calcium phosphate particles, hydroxide apatite particles, carbonate apatite particles, fullerenes, carbon nanotubes, and carbon nanohorns. As a method of supporting the carrier on a carrier whose surface is not positively charged, the surface of the carrier particles is modified with a substance having a positive charge, for example, polyethyleneimine, chitosan, polylysine, etc. There are methods such as electrically bonding or introducing a maleimide group on the surface of the carrier particle, while introducing a thiol group at the end of the linear double-stranded CpG ODN to form a covalent bond.

The weight ratio of the double-stranded CpG ODN to the carrier is preferably adjusted as appropriate according to the base chain length of the double-stranded CpG ODN, the properties of the carrier, the antigen used together, and the like. Typically, the weight ratio of the double-stranded CpG ODN: carrier is 0.05: 1 to 10: 1.

The effect of the oligonucleotide complex is on cells carrying TLR9, such as human and mouse plasmacytoid dendritic cells, mouse macrophages, mouse stationary dendritic cells, and then on type I IFN and inflammatory cytokine genes. It can be determined by quantifying the expression level or by quantifying the secretion amount of type I IFN and inflammatory cytokines. For example, the gene expression level can be quantified by real-time quantitative PCR or the like, and the secretion amount can be quantified by the ELISA method or the like.

The complex can be expected to exert an adjuvant effect when given together with an antigen or an allergen. The complex can be administered in admixture with a free antigen or allergen. Further, there is a method in which the antigen or allergen is complexed to the same carrier as the complex and administered, or a method in which the antigen or allergen is previously bound to the double-stranded CpG ODN and then complexed to the carrier for administration. Further, there is a method in which an antigen or an allergen is complexed with a carrier different from the complex, mixed with the complex, and administered.

The method of complexing the antigen or allergen with the carrier is not particularly limited. For example, after encapsulating the antigen or allergen in the hollow of the cationic liposome, a linear double-stranded CpG ODN is electrostatically applied to the surface of the cationic liposome. There are a method of adsorbing a linear double-stranded CpG ODN and an antigen or an allergen on the surface of calcium phosphate particles, hydroxyapatite particles, and carbonate apatite particles at the same time.

In addition, by mixing linear double-stranded CpG ODN with an antigen or allergen in the raw material liquid for preparing calcium phosphate particles, hydroxide apatite particles, and carbonate apatite particles, two linear particles are contained in these particles. The chain CpG ODN and the antigen or allergen can be included at the same time. When an antigen or allergen is bound to a linear double-stranded CpG ODN in advance and then supported on a carrier, the terminal of the linear double-stranded CpG ODN is modified with a thiol group, while the amino group of the antigen or allergen is used. There is a method of introducing a maleimide group and covalently bonding a thiol group and a maleimide group.

Examples of the antigen include hand-foot-and-mouth disease virus antigen, dengue virus antigen, West Nile fever virus antigen and the like. Examples of allergens include cedar pollen allergens, ragweed allergens, rice allergens, and mite allergens. In addition, antigens of cancer cells and the like can also be mentioned.

Vaccines refer to medicines used to prevent infectious diseases. By administering a detoxified or attenuated antigen, the body promotes the production of antibodies against pathogens and acquires immunity against infectious diseases. Although not particularly limited, live vaccines using microorganisms and viruses with weakened toxicity include BCG, oral live polio (OPV) vaccine, sputum seedlings (natural pox), measles vaccine, rubella vaccine, measles / rubella mixed vaccine (MR vaccine). ), Mumps, vaccine varicella vaccine, yellow fever vaccine, rotavirus vaccine, herpes zoster vaccine, etc. have been put into practical use. Inactivated vaccines including those obtained by culturing only the virus, bacteria, liquettia, or antigen part that died due to chemical treatment include influenza virus vaccine, pneumonia vaccine, mad dog disease vaccine, cholera vaccine, and DPT vaccine. (Difteria / tetanus mixed vaccine), DPT vaccine (difteria / pertussis / tetanus mixed vaccine), quadruple (DPT-IPV) vaccine (difteria / pertussis / tetanus / inactivated polio mixed vaccine), Japanese encephalitis vaccine , DPT vaccine, etc. have been put into practical use.

Cancer vaccine refers to a vaccine used for the treatment or prevention of cancer. As the antigen (cancer antigen), the full length or a part (peptide) of the antigen protein that is not expressed at all in normal cells or is expressed in a small amount even if it is expressed in normal cells and is overexpressed in cancer cells is used. Although not particularly limited, MAGE in malignant melanoma, HER2 / neu in breast cancer, CEA in colorectal cancer, WT1 in various leukemias and various cancers, NY-ESO in malignant melanoma, esophageal cancer, gastric cancer, ovarian cancer, etc. -1 etc. can be mentioned. These cancer antigen proteins (or their degradation peptides) are recognized by cytotoxic T cells (CTL) in the body and attack cancer cells (cell-mediated immunity). Cancer vaccine therapy treats cancer by artificially administering a cancer antigen (peptide) and inducing (differentiating and proliferating) specific CTLs.
Vaccines aimed at preventing carcinogenic virus infection are also included in cancer vaccines. Hepatitis B virus vaccine (which causes liver cancer through cirrhosis) and human papillomavirus (which causes cervical cancer) have been put into practical use.

In patients with pollinosis and house dust allergies, the helper 2T (Th2) pathway specific to each allergen predominates over the helper 1T (Th1) pathway. That is, one of the therapeutic strategies is to activate the allergen-specific Th1 pathway and make it dominant over the Th2 pathway. In mice, the IgG2a / IgG1 ratio is an index of the Th1 / Th2 ratio, but in humans, the IgG4 / IgG1 ratio may be an index of the Th1 / Th2 ratio.

The treatment targets according to the present application include mammals including humans (primates such as monkeys; companion animals such as dogs and cats; domestic animals such as horses, pigs, cows, goats and sheep; experimental animals such as rats and mice), and birds. (Wild birds or poultry such as chickens and turkeys) or fish (fish-cultured species: freshwater fish such as ayu and salmon, and saltwater fish such as bristle and campachi), but TLR9 is expressed. If it is a species, it does not have to be particularly limited.

Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to these Examples.
<Preparation of single-strand CpG ODN and linear double-stranded CpG ODN>
A double-stranded CpG ODN having each sequence shown in Table 2 and a single-stranded ODN having a sequence complementary to the base sequence were synthesized and hybridized to prepare a double-stranded CpG ODN. Hybridization was performed by using the single-stranded CpG ODN of each SEQ ID NO: shown in Table 2 and the complementary single-stranded CpG ODN in a TES buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA and 0.25 mM NaCl). The mixture was mixed in an equimolar ratio, incubated at 95 ° C. for 10 minutes, and then lowered to 30 ° C. over 60 minutes. This hybridization gave a complete linear double-stranded ODN.

For comparison, the single-stranded and double-stranded ODNs of each SEQ ID NO: shown in Table 3 and for reference, the single-stranded and double-stranded ODNs of each SEQ ID NO: shown in Table 4 are the same as above. It was prepared according to the procedure of. B24-PT (SEQ ID NO: 5) in Table 3 is the same as ODN2006 in Table 1.

Single-stranded ODN B24-PT (SEQ ID NO: 5), K3-PT (SEQ ID NO: 8), B24-PD (SEQ ID NO: 1), K3-PD (SEQ ID NO: 4), CpG-free24-PD (SEQ ID NO: 4) 9), and these linear double-stranded ODNs are electrostatically mixed with the cationic liposome Lipofectamine® 2000 (Life Technologies) so that the weight ratio is 1: 1. Combined with.
As a cell containing TLR9, RAW264.7, which is a mouse macrophage cell line, was used. The cell lines were seeded in Eagle's minimal essential medium (MEM) at a density of 3.3 x 10 ⁵ cells / ml. After 24 hours, B24-PT, K3-PT, B24-PD, K3-PD, CpG-free24-PD, and a complex of these double-stranded ODN and lipofectamine 2000, were added to the concentration of lipofectamine 2000. It was added to 5 μg / ml. That is, the ODN (B24-PT, K3-PT, B24-PD, K3-PD, and their double-stranded DNA) bound to Lipofectamine 2000 was added at a concentration of 5 μg / ml. Become. After 6 hours, the cells were collected, total RNA was extracted with ISOGEN (manufactured by Japan Gene), and then cDNA was synthesized with reverse transcriptase (TaKaRa Bio). Using this cDNA as a template, the expression level of the IFN-β gene was measured by real-time quantitative PCR. The expression level of the IFN-β gene was normalized by the expression level of the GAPDH gene. Primer sequences for measuring IFN-β expression by real-time quantitative PCR are forward, 5'-GGTCCGAGCAGAGATCTTCA-3'(SEQ ID NO: 29); reverse, 5'-TCACTACCAGTCCCAGAGTCC-3' (SEQ ID NO: 30), GAPDH gene expression. The primer sequence for quantification was forward, 5'-GTGGACCTCATGGCCTACAT-3'(SEQ ID NO: 31); reverse, 5'-TGTGAGGGAGATGCTCAGTG-3' (SEQ ID NO: 32).

As shown in FIG. 1, no induction of IFN-β was observed in either the single-stranded or double-stranded complex of B24-PT and K3-PT with lipofectamine 2000. On the other hand, in B24-PD and K3-PD, significantly higher IFN-β was induced in the double strand. In addition, double-stranded B24-PD had higher IFN-β-inducing ability than double-stranded K3-PD.

As described above, in Example 1, high IFN-β induction via TLR9 from RAW264.7 cells was observed in the complex of double-stranded B24-PD and lipofectamine 2000. Double-stranded B24-PD is 24 base pairs and contains four CpG in each strand. The double-stranded B48-PD, which is a combination of two double-stranded B24-PDs, is 48 base pairs and contains eight CpG in each strand. In addition, the double-stranded B72-PD in which three double-stranded B24-PDs are connected is 72 base pairs and contains 12 CpG in each strand. These double-stranded CpG ODNs were bound to Lipofectamine 2000 in the same manner as in Example 1 to stimulate RAW264.7 cells.

The results are shown in FIG. In FIG. 2, "ds" indicates that it is a double strand, "PT" is a phosphothioate skeleton, and "PD" is a phosphodiester skeleton. As shown in FIG. 2, the amount of IFN-β induction by the double-stranded B48-PD was significantly higher than that of the double-stranded B24-PD. However, the amount of IFN-β induction by double-stranded B72-PD was the same as that of double-stranded B48-PD. From this, it was suggested that the high IFN-β induction amount and the number of CpG sequences are not in a simple proportional relationship, but that there is an optimum number. On the other hand, phosphorothioated double-stranded B72-PT hardly induced IFN-β. This means that in phosphorothioated double-stranded CpG ODN, increasing the number of CpG does not improve the IFN-β inducibility.

RAW264.7 cells have cytoplasmic DNA receptors such as DAI, IFI16, DDX41 and cGAS as well as TLR9. The cytoplasmic DNA receptor recognizes double-stranded DNA and induces type I IFN regardless of the base sequence. Therefore, IFN-β by double-stranded B24-PD, double-stranded B48-PD and double-stranded B72-PD is considered to be induced via both TLR9 and cytoplasmic DNA receptors. Therefore, IFN-β induction from CpG-free double-stranded CpG-free24-PD, double-stranded CpG-free48-PD, and double-stranded B72-PD at 24 base pairs, 48 base pairs, and 72 base pairs, respectively. I checked the amount.

As shown in FIG. 2, from CpG-free double-stranded CpG-free24-PD, double-stranded CpG-free48-PD, and double-stranded B72-PD, which are 24 base pairs, 48 base pairs, and 72 base pairs, respectively. The amount of IFN-β induction was significantly lower than that of double-stranded CpG-free24-PD, double-stranded B48-PD and double-stranded B72-PD. From this, it was found that most of the IFN-β induction from double-stranded CpG-free24-PD, double-stranded B48-PD and double-stranded B72-PD is mediated by TLR9.

Under the same conditions as in Example 2, the amount of induction of IL-12, which is an inflammatory cytokine, was measured by real-time quantitative PCR. The primer sequence for IL-12 induction measurement was forward, 5'-GAAAGGCTGGGTATCGG-3'(SEQ ID NO: 33); reverse, 5'-GGCTGTCCTCAAACTCAC-3' (SEQ ID NO: 34). As shown in FIG. 3, double-stranded B24-PD, double-stranded B48-PD, and the complex of double-stranded B72-PD and lipofectamine 2000 all induce IL-12 from RAW264.7 cells. did. In addition, unlike IFN-β, the amount of induction was higher in double-stranded B24-PD than in double-stranded B48-PD and double-stranded B72-PD. This result indicates that the phosphodiester double-stranded CpG ODN complex retains not only IFN-β but also IL-12 inducibility.

The IFN-β induction amounts of free double-stranded B72-PD and double-stranded B72-PD, which did not form a complex, and the complex of lipofectamine 2000 with them were compared. The complex of double-stranded B72-PD and lipofectamine 2000 was prepared in the same manner as in Example 1. Culturing of RAW264.7 cells was also carried out in the same manner as in Example 1. Free double-stranded B72-PD, which did not form a complex with Lipofectamine 2000, was added to the medium to a concentration of 50 μg / ml. This concentration is 10 times higher than the concentration of double-stranded B72-PD complexed with Lipofectamine 2000 (5 μg / ml).

As shown in FIG. 4, double-stranded B72-PD induced IFN-β only when complexed with lipofectamine 2000. This means that complexation is essential for IFN-β induction by the double-stranded CpG ODN of phosphodiesters.

Double-stranded B72-PD and double-stranded CpG-free72-PD are electrostatically bound to DOTAP (Roche Life Science), which is a cationic liposome, by mixing them so that the weight ratio is 1: 6. It was. RAW264.7 cells were cultured in the same manner as in Example 1 and added to the medium so that DOTAP had a concentration of 30 μg / ml. That is, the concentration of double-stranded B72-PD and double-stranded CpG-free72-PD complexed with DOTAP in the medium is 5 μg / ml. On the other hand, free double-stranded B72-PD and double-stranded CpG-free72-PD not complexed with DOTAP were added to the medium to a concentration of 50 μg / ml. This concentration is 10 times the concentration of double-stranded B72-PD complexed with DOTAP (5 μg / ml).

As shown in FIG. 5, double-stranded B72-PD complexed with DOTAP also induced IFN-β. Since double-stranded CpG-free72-PD complexed with DOTAP hardly induces IFN-β, IFN-β by double-stranded B72-PD complexed with DOTAP is not a cytoplasmic DNA receptor. It can be seen that it is via TLR9.

FIG. 6 shows a comparison of the IFN-β induction amount of the double-stranded B72-PD complex with DOTAP shown in FIG. 5 and the complex with lipofectamine 2000 shown in FIG. As shown in FIG. 6, the amount of IFN-β induction from the double-stranded B72-PD complexed with DOTAP was significantly lower than that when complexed with Lipofectamine 2000. This indicates that the carrier complexing the double-stranded CpG ODN of the phosphodiester has a significant effect on IFN-β induction.

Single-stranded B72-PD, double-stranded B72-PD and double-stranded CpG-free72-PD were bound to the surface of calcium phosphate particles and TLR9-dependent IFN-β induction from RAW264.7 cells was investigated. 17.52 μl of distilled water was added to 2.48 μl of a 2 mol / l CaCl _{2 solution to make a total of 20 μl.} 12.5 μl of this solution was added to 100 μl of 2 x Hank's Balanced Salt Solution (manufactured by Life Technologies) to obtain a calcium phosphate precipitate. This calcium phosphate precipitate consisted of rod-shaped particles about 150 nm in length. The obtained calcium phosphate particles (about 11.6 μg) were adsorbed with 5 μg each of single-stranded B72-PD, double-stranded B72-PD, and double-stranded CpG-free72-PD. This was added to RAW264.7 cells so that the ODN concentration was 5 μg / ml, and the amount of IFN-β induced was measured 6 hours later.

As shown in FIG. 7, the double-stranded B72-PD complexed with the calcium phosphate particles showed a significantly higher IFN-β inducibility than the single-stranded B72-PD. In addition, since double-stranded CpG-free72-PD complexed with calcium phosphate particles hardly induces IFN-β, IFN-β complexed with calcium phosphate particles is cytoplasmic. It can be seen that it is mediated by TLR9, not by the DNA receptor. Furthermore, this result indicates that IFN-β can be induced by binding phosphodiester linear double-stranded CpG ODN to particles other than cationic liposomes.

Human peripheral blood mononuclear cells (Cellular Technology Limited., OH, USA) purchased from Cellular Technology Limited (OH, USA) are collected by centrifugation and suspended in 800 μl of autoMACS Rincing Solution (Militenyi Biotech, Bergisch Gladbach, Germany). After that, 200 μl of CD14 MicroBeads (Militenyi Biotech) was added and incubated at 6 ° C. for 15 minutes. The Micro Beads were collected by centrifugation, suspended in 1000 μl of autoMACS Rincing Solution, and then passed through an LS column (Militenyi Biotech) under a magnetic field (MidiMACS Separation Unit, Militenyi Biotech). CD14 ⁺ monocytes were recovered from this column. ^{From the eluate of this column, CD304 +} plasmacytoid dendritic cells and CD20 ⁺ B cells were isolated using CD304 MicroBeads (Militenyi Biotech) and CD20 MicroBeads (Militenyi Biotech) by the same method as described above. The proportions of isolated B cells, plasmacytoid dendritic cells and monocytes in human peripheral blood mononuclear cells were 1.2-2.0%, 0.1-0.63% and 12-20%, respectively.
B cells and plasmacytoid dendritic cells isolated from human peripheral blood mononuclear cells were seeded on a ^{96-well flat-bottom plate to a density of 2x10 4 cells / well.} These cells were prepared in 200 μl RPMI 1640 (Invitrogen, Life Technologies, CA, USA) with 10% (v / v) FBS, 1 ml L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin. It was cultured.
B24-PT, B72-PT, B72-PD, CpG-free72-PD, and their double-stranded DNA were complexed with DOTAP in the same manner as in Example 5 to seed plasmacytoid dendritic cells. Added at the same time. After 48 hours, the amount of IFN-α induced was measured by the IFN-α Enzyme-linked immunosorbent assay kit (IFN-α ELISA kit, Affymetrix, CA, USA).

As shown in FIG. 8, in B24-PT and B72-PT, the amount of IFN-α induction does not change significantly even if it is double-stranded, but in B72-PD, it is compared with single-strand by making it double-stranded. The amount of IFN-α induction increased remarkably. In addition, since there is no IFN-α induction from double-stranded CpG-free72-PD, it means that IFN-α from the complex of double-stranded B72-PD and DOTAP is dependent on TLR9. There is.

Next, B24-PT, B72-PD, CpG-free72-PD, and their double-stranded DNA were complexed with DOTAP in the same manner as in Example 1 and added at the same time as B cell seeding. In addition, free B24-PT, B72-PD, CpG-free72-PD, which were not complexed with DOTAP, and their double-stranded DNA were added to the medium at a concentration of 50 μg / ml. After 48 hours, the amount of IL-6 induced was measured by IL-6 ELISA kit (Affymetrix).

As shown in FIG. 9, free single-strand B24-PT and single-strand B72-PD, which were not complexed with DOTAP, both induced IL-6. However, in the double strand, B24-PT induced IL-6, but B24-PD did not. In the complex with DOTAP, single-stranded B72-PD induced IL-6, but double-stranded B72-PD did not. On the other hand, the complex of double-stranded B24-PT and DOTAP induced IL-6. The lack of ability to induce inflammatory cytokines like IL-6 in double-stranded B72-PD suggests that the side effects of double-stranded CpG ODN of phosphodiesters are small.

B24-PT, B72-PT, B72-PD, CpG-free72-PD, and their double-stranded DNA were complexed with DOTAP in the same manner as in Example 1 and added at the same time as monocyte seeding. After 48 hours, the amount of IFN-α induced was measured.

As shown in FIG. 10, double-stranded B72-PD induced IFN-α. Since human monocytes do not have TLR9 and double-stranded CpG-free72-PD also induces IFN-α, the induction of IFN-α from double-stranded B72-PD is mediated by the cytoplasmic DNA receptor. it is conceivable that. This indicates that a double-stranded CpG ODN complex of phosphodiesters, such as double-stranded B72-PD, also induces IFN-α when taken up by cells lacking TLR9.

When Lipofectamine 2000 was dispersed in OptiMEM (Thermo Fisher Scientific Ltd.) at a concentration of 50 μg / ml and the size was measured with a dynamic light scattering meter, the size increased with the passage of time as shown in FIG. Next, complexes of various sizes of lipofectamine 2000 and ds B72-PD (double strand of SEQ ID NO: 3) with different lapses of time were prepared by the method described in Example 1. The size of the complex formed was stable and was about the same as the size of the original Lipofectamine 2000. RAW264.7 cells were stimulated by the method described in Example 1 with these complexes of different sizes. Figure 12 shows the relationship between the size of the complex given to RAW264.7 cells and the amount of IFN-β induced 6 hours after the addition of the complex. The complex of lipofectamine 2000 and double-stranded B72-PD showed high IFN-β inducibility above 700 nm.

Double-stranded ds B72-PD was prepared by the method described in Example 1 using B72-PD of SEQ ID NO: 3 in Table 2 and a single-stranded ODN completely complementary thereto. The ds B72-PD and Lipofectamine 2000 were mixed in a weight ratio of 1: 2 and 1: 1 to obtain complexes having sizes of 737 ± 213 nm and 692 ± 129 nm, respectively. IFN- ^{when RAW264.7 cells seeded at a cell density of 3.4x10 5} cells / ml and cultured for 16 hours were stimulated with these complexes for 6 hours so that the concentration of lipofectamine 2000 was 5 μg / ml. The β-induction amount is shown in FIG. The amount of IFN-β induction was similar in both complexes. From this result, it can be seen that when the concentration of lipofectamine 2000 in the culture medium is 5 μg / ml, the binding amount of ds B72-PD should be 0.5 or more with respect to the weight of lipofectamine 2000.

Double-stranded ds B72-PD was prepared by the method described in Example 1 using B72-PD of SEQ ID NO: 3 in Table 2 and a single-stranded CpG ODN completely complementary thereto. This ds B72-PD and Lipofectamine 2000 were mixed so as to have a weight ratio of 1: 2 to form a complex. The size of this complex was 737 ± 213 nm. This complex was added to RAW264.7 cells seeded at a cell density of 3.4x10 ⁵ cells / ml and cultured for 16 hours so that the concentration of lipofectamine 2000 was 1, 2 and 5 μg / ml for 6 hours. Later, the amount of IFN-β induction was measured. As shown in FIG. 14, as the amount of the complex added increased, the amount of IFN-β induced also increased.

A 72-base single-stranded CpG ODN composed of the phosphodiesters shown in Table 5 and a single-stranded ODN having a sequence complementary to the base sequence were synthesized, and the double-stranded CpG ODN was prepared by the method described in Example 1. Prepared. The CpG ODNs of SEQ ID NOs: 18 to 22 shown in Table 5 are partially modified base sequences of B72-PD of Table 2 SEQ ID NO: 3, and the modified bases are double underlined. Table 5 B72M1-PD of SEQ ID NO: 18 differs from B72-PD of Table 2 SEQ ID NO: 3 in five nucleotide sequences. Table 5 B72M2-PD of SEQ ID NO: 19 differs from B72-PD of Table 2 SEQ ID NO: 3 in seven nucleotide sequences. Table 5 B72M3-PD and B72M4-PD of SEQ ID NO: 20 and SEQ ID NO: 21 differ in 12 base sequences from B72-PD of Table 2 SEQ ID NO: 3. Table 5 B72M5-PD of SEQ ID NO: 22 differs from B72-PD of Table 2 SEQ ID NO: 3 in 14 nucleotide sequences.

Preparation of a complex of these double-stranded CpG ODN and lipofectamine 2000 and stimulation of RAW264.7 cells with these complex were performed by the method described in Example 1. The results are shown in Fig. 15. The complex of any of the double-stranded CpG ODNs shown in Table 5 and lipofectamine 2000 showed high IFN-β inducibility as in the complex of ds B72-PD and lipofectamine. In addition, most of the IFN-β induction was due to the fact that these double-stranded CpG ODN complexes induced significantly higher IFN-β than the CpG-free double-stranded CpG-free72-PD complex. It turns out that it depends on TLR9.

Next, a complex of these double-stranded CpG ODN and DOTAP was prepared by the method described in Example 5, and RAW264.7 cells were stimulated with these complex. The results are shown in Fig. 16. Any of the double-stranded CpG ODN and DOTAP complexes shown in Table 5 showed IFN-β inducibility equal to or higher than that of the ds B72-PD and DOTAP complex.

A 48-base single-stranded CpG ODN composed of the phosphodiesters shown in Table 6 and a single-stranded ODN having a sequence complementary to the base sequence were synthesized, and the double-stranded CpG ODN was prepared by the method described in Example 1. Prepared. The CpG ODNs of SEQ ID NOs: 23 to 27 shown in Table 6 are partially modified base sequences of B48-PD of Table 2 SEQ ID NO: 2, and the modified bases are double underlined. Table 6 B48M1-PD of SEQ ID NO: 23 differs from B48-PD of Table 2 SEQ ID NO: 2 in five base sequences. Table 6 B48M2-PD of SEQ ID NO: 24 differs from B48-PD of Table 2 SEQ ID NO: 2 in seven nucleotide sequences. B48M3-PD and B48M4-PD of SEQ ID NO: 25 and SEQ ID NO: 26 in Table 6 differ in 12 base sequences from B48-PD in Table 2 SEQ ID NO: 2. Table 6 B48M5-PD of SEQ ID NO: 27 differs from B48-PD of Table 2 SEQ ID NO: 2 by 14 nucleotide sequences. CpG48-PD of SEQ ID NO: 28 in Table 6 contains 10 CpGs, and the sequences of the two bases before and after CpG are different from those of B48-PD in SEQ ID NO: 2 in Table 2 and SEQ ID NOs: 23 to 27 in Table 6.

Preparation of a complex of these double-stranded CpG ODN and lipofectamine 2000 and stimulation of RAW264.7 cells with these complex were performed by the method described in Example 1. The results are shown in FIG. It can be seen that ds B48M1-PD, ds 48M2-PD and ds B48M3-PD have a lower amount of IFN-β inducing than ds B48-PD, but still have a high level of IFN-β inducing ability. In addition, ds B48M4-PD, ds B48M5-PD and ds CpG48-PD further reduced the ability to induce IFN-β, but since it was higher than ds CpG-free72-PD, the induction of IFN-β by these also resulted in TLR9. You can see that it is through.

Double-stranded ds B72-PD was prepared by the method described in Example 1 using B72-PD of SEQ ID NO: 3 in Table 2 and a single-stranded ODN completely complementary thereto. This ds B72-PD and Lipofectamine 2000 were mixed so as to have a weight ratio of 1: 1 to form a complex. This complex solution was concentrated 10-fold with an Amicon Ultra 0.5 mL Centrifugal Filter (Merck Millipore, Darmstadt, Germany) to prepare a complex solution containing 50 μg of ds B72-PD in 100 μl. That is, 100 μl of this concentrated solution contains a complex in which 50 μg of ds B72-PD is electrostatically bound to 50 μg of lipofectamine 2000. A 100 μl solution containing 50 μg of ds B72-PD and a 100 μl solution containing 200 μg of model antigen ovalbumin (OVA) were mixed and administered subcutaneously to the back of 6 6-week-old mice (C57BL / 6J). The ds B72-PD complex and OVA prepared in the same manner were administered subcutaneously to the back of the mouse 7 days later. Blood was collected from the posterior vena cava under influenza inhalation anesthesia 7 days after the second administration, that is, 14 days after the first administration. A part of the collected blood was treated with EDTA, and the rest was centrifuged, and then serum was collected. After hemolysis and fixation treatment, EDTA-treated blood is reacted with PE-Cy5 hamster anti-mouse CD3 antibody, FITC-rat anti-mouse CD8 antibody, and H2-Kb SIINFEKL Class I iTAgTM MHC tetramer, and is OVA-specific by FACS. The proportion of CD8-positive T cells was examined. OVA-specific IgG1, OVA-specific IgG2a and OVA-specific IgE contained in serum were quantified by ELISA. The proportion of OVA-specific CD8-positive T cells and the production of OVA-specific IgG1, OVA-specific IgG2a and OVA-specific IgE were as follows: 6 animals in which 100 μl solution containing 200 μg OVA was subcutaneously administered to the back twice every 7 days. It was compared with mice and 6 untreated mice.

The results of the proportion of OVA-specific CD8-positive T cells are shown in FIG. The ratio of OVA-specific CD8-positive T cells to CD8-positive T cells in the blood in the group of mice treated with the ds B72-PD complex and OVA was significantly higher than that of the group of mice treated with OVA alone, and ds B72- The effect of inducing antigen-specific CD8-positive T cells of the PD complex was shown. On the other hand, no significant increase in the proportion of OVA-specific CD8-positive T cells was observed in mice administered with OVA alone, indicating that it is difficult to induce antigen-specific CD8-positive T cells with antigen-only administration. Was done.

The amount of OVA-specific IgG1 antibody produced in serum is shown in FIG. The amount of OVA-specific IgG1 antibody produced was also significantly increased in the group of mice to which OVA alone was administered. In the group of mice administered with the ds B72-PD complex and OVA, the amount of OVA-specific IgG1 antibody produced was significantly increased as compared with the group of mice administered with OVA alone.

The amount of OVA-specific IgG2a antibody produced in serum is shown in FIG. Regarding the production amount of OVA-specific IgG2a antibody, the production amount of OVA-specific IgG2a antibody was significantly increased in the group of mice administered with the ds B72-PD complex and OVA than in the group of mice administered with OVA alone. .. On the other hand, in the mouse group to which only OVA was administered, no significant increase in OVA-specific IgG2a was observed, indicating that it is difficult to induce OVA-specific IgG2a by administration of antigen alone.

The amount of OVA-specific IgE antibody produced in serum is shown in FIG. In the group of mice administered with the ds B72-PD complex and OVA, the mean value of the production of OVA-specific IgE antibody was increased as compared with the group of mice administered with OVA alone, but no significant increase was observed.

IgG is involved in a delayed immune response and IgE is involved in a fast-acting immune response. Therefore, the ds B72-PD complex can enhance the antigenicity when administered together with the antigen, and can be used to enhance its effect when used in combination with the vaccine antigen. On the other hand, it does not affect the immediate immune response (anaphylaxis, etc.), suggesting that it is highly safe.

Human peripheral blood mononuclear cells (Cellular Technology Limited., OH, USA) were ^{seeded on a 96-well flat-bottom plate at a density of 1x10 6} cells / well and bound to DOTAP by electrostatic interaction. ds B72-PD, ds CpG-free72-PD, ODN2216 (A2216 in the figure), and B24-PT were added to a concentration of 5 μg / ml (DOTAP concentration is 30 μg / ml). These cells were cultured in RPMI 1640 with 10% (v / v) FBS, 10 mM HEPES, and 2 mM L-glutamine. After 6 hours, cells were harvested and reacted with PerCP / Cy5.5 anti-human CD303 (BDCA-2), FITC anti-human CD14, Alexa Fluor 700 anti-human CD20, PE anti-human CD80 for 15 minutes at room temperature. After washing with PBS, the proportion of CD80-positive cells expressing the co-stimulator CD80 was analyzed with a cell analyzer (SP6800, SONY).

The proportion of CD80-positive cells in human peripheral blood mononuclear cells is shown in FIG. The proportion of CD80-positive cells in each DOTAP complex was increased compared to untreated cells. The proportion of CD80-positive cells in the ds B72-PD complex was similar to that of the CpG-A A2216-DOTAP complex. The proportion of CD80-positive cells in the ds CpG-free72-PD complex was lower than that of the ds B72-PD complex or the A2216 complex. An increase in the proportion of CD80-positive cells was also observed in the B24-PT complex.

In human peripheral blood mononuclear cells, the main cell types expressing CD80 are considered to be B cells, monocytes, and dendritic cells, so CD20-positive cells (mainly B cells) and CD14-positive cells ( The proportion of CD80-positive cells among mainly monocytes) and CD303-positive cells (mainly dendritic cells) was examined. The results are shown in Table 7. The proportion of CD80-positive cells among CD20-positive cells was highest in the ds B72-PD complex, suggesting that the ds B72-PD complex activates B cells, but the ds CpG-free72-PD complex. The proportion of CD80-positive cells in the cells was not significantly increased as compared with the untreated cells. The proportion of CD80-positive cells in CD14-positive cells was highest in the A2216 complex, but a significant increase in CD80-positive cells was also observed in the ds B72-PD complex. Furthermore, in the proportion of CD80-positive cells among CD303-positive cells, high CD80-inducing ability was observed in all the complexes, but the ds B72-PD complex showed the highest inducible ability.

Two types of signals are required for antigen-presenting cells to initiate an immune response. The first signal is an antigen-specific signal mediated by a T cell receptor, and the second signal is a non-antigen-specific signal mediated by a co-stimulating molecule. The ds B72-PD complex efficiently induces the co-stimulating molecule CD80, suggesting that the ds B72-PD complex is effective as an antigenic enhancer (adjuvant).

A complex of lipofectamine 2000 and ds B72-PD, ds CpG-free72-PD, and the plasmid vector pAcGFP-N1 (TaKaRa Bio, Siga, Japan) was prepared by the method described in Example 1, and each complex was prepared. Stimulated RAW264.7 cells with. The result of measuring the IFN-β induction amount after 6 hours is shown in FIG. 23, and the result of measuring the IL-12 induction amount is shown in FIG. 24. Both IFN-β and IL-12 were significantly more inducible by the ds B72-PD complex than the pAcGFP-N1 complex. The 4700 base pair pAcGFP-N1 is a phosphodiester double-stranded circular DNA containing CpG, whereas the linear ds B72-PD complex has higher IFN-β inducibility and IL than the circular double-stranded DNA. It was shown to have -12 inducibility.

The double-stranded oligonucleotide complex of the present invention has high TLR9-mediated type I IFN-inducing activity and low inflammatory cytokine-inducing activity. The complex has no concern about side effects due to phosphorothioate, is easy to prepare, and is useful as a therapeutic agent for allergies, vaccine adjuvants, and the like.

Claims (21)

It is a linear double-stranded oligonucleotide containing 10 to 100 base pairs, and each single-stranded oligonucleotide constituting the double strand has a phosphodiester-mediated citocin-guanine sequence (CpG) of 2 to 20. pieces comprises 90% or more phosphodiester linkages der bonds between nucleotides of the single-stranded oligonucleotide is, the single-stranded oligonucleotide is 1-3 bases other than CpG in the following nucleotide sequence and sequence-deficient, that having a sequence of any of a substituted or added in the sequence, double-stranded oligonucleotide:
5'-TCGTCGTTTTGTCGTTTTGTCGTTTCGTCGTTTTGTCGTTTTGTCGTT-3'(SEQ ID NO: 2)
5'-TCGTCGTTTTGTCGTTTTGTCGTTTCGTCGTTTTGTCGTTTTGTCGTTTCGTCGTTTTGTCGTTTTGTCGTT-3'(SEQ ID NO: 3) . The double-stranded oligonucleotide according to claim 1, wherein each single-stranded oligonucleotide does not contain a palindrome sequence. The double-stranded oligonucleotide according to claim 1 or 2, wherein the bonds between the nucleotides of each single-stranded oligonucleotide are all phosphodiester bonds. The double-stranded oligonucleotide according to any one of claims 1 to 3, wherein the single-stranded oligonucleotide has any of the following sequences.
5'-TCGTCGTTTTGTCGTTTTGTCGTTTCGTCGTTTTGTCGTTTTGTCGTT-3'(SEQ ID NO: 2)
5'-TCGTCGTTTTGTCGTTTTGTCGTTTCGTCGTTTTGTCGTTTTGTCGTTTCGTCGTTTTGTCGTTTTGTCGTT-3'(SEQ ID NO: 3) An immunostimulating oligonucleotide complex comprising a carrier and the double-stranded oligonucleotide according to any one of claims 1 to 4, which is complexed with the carrier. The immunostimulating oligonucleotide complex according to claim 5, wherein the carrier is selected from liposomes, polymeric compounds, and inorganic compounds. The immunostimulating oligonucleotide complex according to claim 5 or 6, wherein the average particle size is 100 nm or more. The immunostimulating oligonucleotide complex according to claim 5 or 6, wherein the weight ratio of the double-stranded oligonucleotide to the carrier is 0.05: 1 to 10: 1. A vaccine adjuvant comprising the immunostimulatory oligonucleotide complex according to any one of claims 5 to 8. The immunostimulatory oligonucleotide complex according to any one of claims 5 to 8 for use in preventing infectious diseases of mammals including humans, birds or fish, and is nontoxic from a pathogen causing an infectious disease. An immunostimulatory oligonucleotide complex for promoting the production of an antibody against a pathogen in the body and acquiring immunity against an infectious disease by continuously or simultaneously administering the attenuated or attenuated antigen. Use of the immunostimulatory oligonucleotide complex according to any one of claims 5 to 8 in the production of a vaccine for preventing infectious diseases in mammals including humans, birds or fish. The immunostimulatory oligonucleotide complex according to any one of claims 5 to 8, for the prevention of infectious diseases in mammals including humans, birds or fish. The immunostimulatory oligonucleotide complex according to any one of claims 5 to 8 for use in the treatment or prevention of cancer.
By continuously or simultaneously administering the cancer antigen or a part thereof and the immunostimulatory oligonucleotide complex according to any one of claims 5 to 8, cytotoxic T cells against the cancer antigen (cell-damaging T cells) ( An immunostimulatory oligonucleotide complex that treats or prevents cancer by inducing CTL) and attacking cancer cells that present cancer antigens. Use of the immunostimulatory oligonucleotide complex according to any one of claims 5 to 8 in the production of a vaccine for treating or preventing cancer. The immunostimulatory oligonucleotide complex according to any one of claims 5 to 8 for treating or preventing cancer. A pharmaceutical composition for treating or preventing allergies, which comprises the immunostimulatory oligonucleotide complex according to any one of claims 5 to 8. The pharmaceutical composition according to claim 16, further comprising an allergen or a part thereof. The immunostimulatory oligonucleotide complex according to any one of claims 5 to 8 for the treatment or prevention of allergies.
An immunostimulatory oligonucleotide complex for treating or preventing allergies by activating allergen-specific helper 1T (Th1) cells more than helper 2T (Th2) cells. The immunostimulating oligonucleotide complex according to claim 18, further for administering the allergen or a part thereof continuously or simultaneously with the immunostimulating oligonucleotide complex . Use of the epidemic oligonucleotide complex according to any one of claims 5 to 8 in the manufacture of a pharmaceutical for treating or preventing allergies. The immunostimulatory oligonucleotide complex according to any one of claims 5 to 8 for treating or preventing allergies.